In recent years, significant research efforts have been made worldwide to develop transportation fuels and industrial chemicals from sustainable biomass to effectively deal with the depletion of fossil fuel and global warming caused by greenhouse gases.
Biomass comprises lignocellulosic biomass, which mainly includes phanerophytes, and algal biomass, which mainly includes algae growing in water. Cellulose, one of the structural components that constitute biomass, is an abundant resource which takes up 20% to 50% of biomass, and is also a highly polymerized condensation product of glucose, which is considered as a primary nutrient for fermentation strains. Thus, numerous research and development efforts have focused on mass production of highly purified glucose from cellulose.
In addition to cellulose, however, lignocellulosic biomass also contains hemi-cellulose (about 15 to 35%), which is prone to over-decomposition, and lignin (about 10 to 30%), which is difficult to be reduced due to its complex structure. Additionally, lignocellulosic biomass also contains water-extractable substances including water-soluble starch, free sugars, proteins, pectin, tannin, various alkaloids, organic acids, various inorganic salts, and the like. Generally, such ingredients are contained slightly less in quantity: 20 to 30% in grassy biomass and 5 to 20% in woody biomass (see Michael E. Himmel (2009) Biomass recalcitrance, Blackwell Publishing; Run-Cang Sun (2010) Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels, Elsevier).
Among the extractable substances contained in lignocellulosic biomass, starch or free sugar can be used in the process of producing fermentable sugars. The rest of the ingredients, however, not only act as impurities but may also deteriorate the rate of sugar yield during the production of fermentable sugars, and thus, it is important to recover or remove these ingredients.
Korean Laid-open Patent Publication No. 2011-0040367 discloses equipment that discharges liquid material, as a process of continuous fractionation of biomass, by introducing hot water into a reactor, stirring the mixture for a certain period, and then discharging a liquid material using vapor pressure. Although this prior art was designed to extract hot water-extractable substances by using the equipment, it was not suitable for removing the extractable substances because the extraction of liquid material through valves does not take place sufficiently under high pressure, and the total recovery rate was merely 50% or lower, which was very low even after repeating the fractionation and recovery processes several times due to the tunneling effect of the contents during the extraction process.
Inbicon A/S discloses a method for removing extractable substances from biomass by subjecting the biomass to autohydrolysis, removing a liquid material containing a large amount of ingredients that inhibits microbial activity via solid-liquid separation, and then washing the pretreated solid residue with water (see Jan Larsen et al., 2012, Biomass and Bioenergy, 46, 36-45). Although this method has an advantage in that it yields clean pretreated solid residues which can be used for production of high quality fermentable sugar, it has a downside that an increase in manufacturing cost is inevitable due to solid-liquid separation and repeated washing processes. Also, an additional process of adding a certain amount of the pretreated liquid material containing microbial growth inhibitors is required to prevent possible contamination which can be caused by lactic acid bacteria during enzymatic saccharification or alcohol fermentation. For this reason, the contents of the resulting product would become more complex after going through a reaction under high temperature and high pressure conditions and, as a result, the resulting product is added with unknown extractable substances from the biomass. Moreover, the liquid material is obtained as a byproduct from the pretreatment of biomass can only be used for production of low-value products, e.g., fertilizers, and cannot be developed as a value-added product due to the presence of xylose and xylan, as well as over-decomposed products of sugar such as furfural, HMF and the like; protein denaturants generated by Maillard reaction at a high temperature of, for example, 190° C.; various organic acids; lignin degradation products; and various inorganic salts.
In the process of manufacturing glucose from biomass, pretreatment process and saccharification process generally take place consecutively because cellulose is not easily converted to glucose when the biomass is merely in a pulverized form. The pretreatment of biomass refers to a process of treating pulverized or crushed biomass in a physicochemical manner so as to make each structural component of the biomass suitable for fractionation. The saccharification of biomass refers to a process of converting a pretreated cellulose, which has become suitable for more efficient hydrolysis because hemicellulose or lignin that surrounds cellulose has been partially or wholly decomposed or dissolved, to glucose in a physicochemical or biochemical manner.
Examples of conventional methods used in the pretreatment of biomass include autohydrolysis (or hydrothermolysis), dilute acid pretreatment, lime pretreatment, ammonia pretreatment (ARP, etc.), steam explosion and etc. The pretreatment process renders cellulose more reactive towards hydrolyzing enzymes by mostly dissolving hemicellulose or lignin contained in the biomass. Not only do the type of biomass and the reaction conditions heavily affect the efficiency of the pretreatment, these factors also change the types and amounts of compounds other than sugar produced in the saccharification process. Recently, among these examples, autohydrolysis is getting more attention because it provides an economic advantage owing to its simple process and also has wide applicability towards various kinds of biomass.
The enzymatic hydrolysis of the pretreated material refers to a process of converting cellulose contained in the pretreated biomass, which has been converted into a form that can easily react with enzymes, to glucose by treating with cellulases. Depending on the pretreatment method selected, the cellulases used in the enzymatic hydrolysis may further include a variety of enzymes, e.g., hemicellulose, amylase, pectinesterase, and the like, to promote hydrolysis reaction.
After the pretreatment and saccharification processes, the sugar-containing materials obtained from lignocellulosic biomass can be further treated in accordance with two major methods. First, there is a method called simultaneous saccharification and co-fermentation in which a saccharified material (hereinafter referred to as “saccharified material”) containing the residue obtained from saccharification process (hereinafter referred to as “hydrolysis residue”), or after conducting saccharification process, the pretreated material containing a small amount of glucose is directly added with fermentation strains and additives, and which is then subjected to fermentation. Currently, this method has been widely used in studies and empirical manufacturing of bio-alcohols. According to the second method, a sugar solution is prepared via solid-liquid separation after the saccharification has been completed, and then such sugar solution is used as a fermentable sugar.
When a sugar solution is prepared from lignocellulosic biomass via physicochemical pretreatment and enzymatic hydrolysis, the sugar solution contains not only monosaccharides such as glucose but also a number of impurities. Representative examples of the impurities include over-decomposed products of sugar such as aldehydes including furfural and hydroxymethylfurfural (hereinafter referred to as “HMF”), organic acids such as levulinic acid and formic acid, and alcohols such as methanol; as well as hydrolysate of hemicellulose including acetic acids, and hydrolysate of lignin including various phenolic compounds. Depending on the kind of fermentation strain, these impurities may act as a microbial growth inhibitor or metabolite production inhibitor. It has been reported that phenolic compounds, i.e., hydrolysate of lignin, contained in a sugar solution is the most powerful microbial inhibitor; furfural and HMF may act as a selective inhibitor depending on their concentrations; and many kinds of acids such as acetic acid exhibit different physiological reaction depending on the strain. Accordingly, attempts have been made to minimize the effects caused by various impurities or to use the saccharified material from biomass directly for microbial fermentation by improving fermentation strain using molecular biology techniques and selecting suitable microbes from new stains. Yeasts, an ethanologen, are known to be the most resistant strain against microbial inhibitors, and recently studies have focused on modifying such strain for manufacturing of bio-alcohols from a lignocellulosic sugar solution.
On the other hand, microbial growth or metabolite production of most of the industrial microbes such as Escherichia coli or Clostridium acetobutylicum can be greatly hindered by certain impurities. Accordingly, various studies have been conducted, e.g., overliming and polymerization using lignin peroxidase, and the like, so as to detoxify microbial inhibitors contained in sugar solutions obtained from lignocellulosic biomass. Also, an attempt has been made to eliminate such inhibitors by applying different chromatography techniques using adsorption and partition (see Villarreal, M. L. M. et al., Enzyme and Microbial Technology, 40, 17-24, 2006).
Meanwhile, in order to minimize the amount of microbial inhibitor contained in sugar solutions, pretreated biomass may be washed prior to enzymatic saccharification. This technique, however, may cause an undesirable microbial contamination, e.g., L. acidophilus, during the enzymatic saccharification or alcohol fermentation process after the pretreated material has been washed, and thus, some of the pretreated liquid material can be added back in order to avoid such contamination. Although this technique is very useful for alcohol fermentation which employs yeast that is resistant to microbial inhibitors because microbial inhibitors such as acetic acid and phenolic compounds are released from the pretreated biomass as the enzymatic saccharification and ethanol fermentation proceed. However, no application utilizing this technique in the manufacturing of fermentable sugar has been reported until now. Also, in case of pretreated biomass having a small average particle diameter due to pulverization or pretreatment, there is a risk of a decrease in the sugar yield since some of the particlized pretreated biomass may be lost during the washing process.